Summary of work: Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes are formed after oxidative stress. High levels of these lesions are strongly associated with the development of cancer and implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is the mitochondrial DNA rather than the nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve the base excision (BER) and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarifying these pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes. Using in vitro assays we are able to examine the repair of different types of lesions and to measure different steps of the pathway. Furthermore, we can measure DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. We and others identified several protein interactions for the core BER enzymes. These protein interactions are physical and functional and together support the "passing of baton" model, in which BER takes place in different steps supported by individual protein interactions that are components of a repair complex, possibly situated at the DNA lesion. We are finding new protein partners in this process, including poly(ADP) ribose polymerase (PARP), which is activated by DNA strand breaks. We are further examining these interactions in a human disorder, Cockayne syndrome (CS), characterized by premature aging. There are deficiencies in the repair of oxidative DNA damage in the nuclear and mitochondrial DNA of CS cells, and this may be the major underlying cause of the disease. We recently demonstrated that the CSB protein, mutated in CS patients, interacts with PARP1 and that these two proteins cooperate in the cellular responses to oxidative stress. One base lesion, 8-oxoG, is of special interest since it causes mutations, if left unrepaired. We have studied the mechanism of repair of this lesion and find that it is repaired mainly via BER and in a mode that is not coupled to transcription. We also find that the major DNA glycosylase for 8-oxoG repair, oxoguanine DNA glycosylase (OGG1), interacts with and can be phosphorylated by the cyclin-dependent kinase cdk4. This post-translational modification modulates OGG1 catalytic activity, suggesting a role for signaling pathways in the response to oxidative DNA damage.